Project Summary/Abstract The proposed investigation will examine ways in which the human metabolome predicts risk of type 2 diabetes (T2D) and also examine relationships between established dietary factors (in particular the Mediterranean dietary pattern), metabolites, and risk of T2D. High-throughput metabolite profiling (metabolomics) techniques have advanced to allow investigators to rapidly and robustly measure the full profile of thousands of small molecule metabolites circulating in human plasma. A subset of these currently known and unknown/novel metabolites are proximal in the pathophysiology of chronic diseases such as T2D, and some are modulated by a healthy diet. In this project, we propose to leverage rich resources available in three large-scale prospective US studies, the Health Professionals Follow-up Study (HPFS), the Nurses? Health Study (NHS), and the VITamin D and OmegA-3 TriaL (VITAL, ~20% African Americans), to evaluate relationships between circulating plasma metabolomic profiles, diet, and risk of T2D (1,800 incident events, 4,900 plasma samples). In Aim 1, we will examine baseline metabolites and metabolomic profiles associated with incident T2D in the HPFS and NHS (N=1,150 T2D matched case-control pairs identified through 2014) by analyzing both candidate and untargeted metabolites. In Aim 2, we will examine relationships between established dietary factors (e.g., Mediterranean diet score), metabolites, and T2D risk. Furthermore, in a subset of 500 study participants with 6-month repeated measures of diet and measured dietary biomarkers (e.g. total energy intake by doubly-labeled water, protein intake by urinary nitrogen, urinary sodium and potassium, and plasma carotenoids and vitamins), we will examine associations between specific dietary factors and metabolites. In Aim 3, we will replicate significant findings from Aims 1 and 2 in the VITAL study (N=650 T2D matched case- control pairs identified from 2011-2017), and conduct a meta-analysis of the three cohorts. In Aim 4, we will examine 10-year changes in the candidate metabolite levels of Aim 1 in relation to subsequent T2D (2000- 2014); these analyses will be performed in N=400 T2D matched case-control pairs from NHS with two blood samples collected 10 years apart. The highly cost-effective two-stage study design (i.e., discovery in HPFS/NHS and replication in VITAL) will reduce the chance of false positive findings and boost statistical power. This is the first study to evaluate long-term changes in plasma metabolite levels in relation to incident T2D. This novel approach mimics a ?natural experiment?, and the proposed analyses will facilitate causal interpretations. The findings could serve as critical first steps for evaluating both known and unknown/novel metabolites as targets for diet and lifestyle modifications for preventive or intervention strategies aimed at reducing the burden of T2D.